The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling.

The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show that PTEN transcription is upregulated by Egr-1 after irradiation in wild-type, but not egr-1-/-, mice in vivo. We found that Egr-1 specifically binds to the PTEN 5' untranslated region, which contains a functional GCGGCGGCG Egr-1-binding site. Inducing Egr-1 by exposing cells to ultraviolet light upregulates expression of PTEN messenger RNA and protein, and leads to apoptosis. egr-1-/- cells, which cannot upregulate PTEN expression after irradiation, are resistant to ultraviolet-light-induced apoptosis. Therefore, Egr-1 can directly regulate PTEN, triggering the initial step in this apoptotic pathway. Loss of Egr-1 expression, which often occurs in human cancers, could deregulate the PTEN gene and contribute to the radiation resistance of some cancer cells.

Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells.

F9 embryonal carcinoma (EC) cells, cultured in suspension in medium containing 5 X 10(-8) M retinoic acid, aggregate and differentiate into embryoid bodies with an outer layer of visceral endoderm cells that synthesize and secrete alphafetoprotein (AFP) (Hogan, B. L. M., A. Taylor, and E. Adamson, 1981, Nature (Lond.). 291:235-237). Here we analyze the formation of the outer layer of cells as a model for epithelial differentiation. Three morphological phases are described, but analyses of cell numbers and the synthetic rates of some proteins, as well as the appearance of markers of visceral endoderm and basement membrane, show that the formation of the outer layer occurs as an orderly progression of multiple events. The markers used to follow the ontogeny of epithelial layer formation include SSEA-1, l, and i blood group antigens, laminin, fibronectin, type IV collagen, cytoskeletal intermediate filament proteins (vimentin, Endo A, and B), and AFP. The onset of epithelium formation occurs between the third and fourth day of culture, but its function is maximally expressed only when it is well organized. We found the rate of AFP secretion to be a measure of the proper alignment and maturity of the epithelium which occurs at the seventh or eighth day. This model of epithelium formation may help to explain how similar processes occur during embryogenesis.

Role of laminin in epithelium formation by F9 aggregates.

The formation and maturation of the outer epithelial layer is essential for maximal alphafetoprotein (AFP) production during differentiation of F9 embryoid bodies in the presence of 5 X 10(-8) M retinoic acid (Grover et al., 1983. J. Cell Biol. 96:1690-1696). The critical phase is between the third and the fourth day when the components of the extracellular matrix organize into a basement membrane. The role of some of these components in the process of epithelium formation and maturation is analyzed in this paper. The role of laminin was investigated by testing the effect of exogenous laminin and antilaminin in cultures of differentiating F9 aggregates. Tests included growth rates, morphological changes, AFP production, determination of AFP mRNA levels, and fluorescent staining for basement membrane components and for epithelial markers. At concentrations greater than 5 micrograms/ml, exogenous laminin inhibited the production of AFP and prevented AFP gene transcription. On the basis of immunofluorescence tests, exogenous laminin appeared to act by preventing the accumulation of a basement membrane and by disrupting the organization of the outer layer into an epithelium. No such effects were produced by fibronectin or collagens type I or IV. Aggregates cultured in the presence of antilaminin also failed to organize an epithelium and did not produce AFP, whereas those in normal rabbit serum differentiated normally. Therefore, endogenous laminin plays a key role not only as a basement membrane structural component but also in organizing the epithelial layer of endoderm cells and hence (indirectly) in gene expression.

UV activates growth factor receptors via reactive oxygen intermediates.

Exposure of mammalian cells to UV irradiation induces rapid and transient expression of early growth response-1 gene (Egr-1) encoding a transcription factor that plays a role in cell survival. These signals from the irradiated cell surface are likely to involve more than one pathway, and we show here that an essential pathway involves activation of several growth factor receptors by reactive oxygen intermediates (ROI). UVC irradiation causes the tyrosine phosphorylation of EGF receptor (EGFR) in mouse NIH 3T3 fibroblasts and HC11 mouse mammary cells. EGFR activation by irradiation of cells is abrogated by suramin, by antioxidants, and by the presence of a dominant negative EGFR. UV induces the formation of complexes between activated EGFR and SOS, Grb2, PLC gamma, and SHC that can be precipitated with antibodies to EGFR. The activation of EGFR by UV is mimicked by H2O2, suggesting that ROI may function upstream of EGFR activation. Our observations support the hypothesis that ROI and growth factor receptors operate in the early steps of the UV signal that lead to the enhanced expression and activity of Egr-1.

Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell.

A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell-substrate adhesion. We have previously shown that neither uvomorulin (E-cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins.

alpha-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.

alphaE-catenin, a cadherin-associated protein, is required for tight junction (TJ) organization, but its role is poorly understood. We transfected an alphaE-catenin-deficient colon carcinoma line with a series of alphaE-catenin mutant constructs. The results showed that the amino acid 326-509 domain of this catenin was required to organize TJs, and its COOH-terminal domain was not essential for this process. The 326-509 internal domain was found to bind vinculin. When an NH2-terminal alphaE-catenin fragment, which is by itself unable to organize the TJ, was fused with the vinculin tail, this chimeric molecule could induce TJ assembly in the alphaE-catenin-deficient cells. In vinculin-null F9 cells, their apical junctional organization was impaired, and this phenotype was rescued by reexpression of vinculin. These results indicate that the alphaE-catenin-vinculin interaction plays a role in the assembly of the apical junctional complex in epithelia.

A network of p73, p53 and Egr1 is required for efficient apoptosis in tumor cells.

p73, a transcription factor rarely mutated in cancer, regulates a subset of p53 target genes that cause cells to respond to genotoxic stress by growth arrest and apoptosis. p73 is produced in two main forms; only TAp73 reiterates the roles of p53, while DeltaNp73 can be oncogenic in character. We show that the TAp73 form produced by TP73 P1 promoter has five distinct Egr1-binding sites, each contributing to the transcriptional upregulation of TAp73 by Egr1 in several cell types. In contrast, TP73 P2 promoter transcribes DeltaNp73, is not induced by Egr1, but is induced by TAp73 and p53. Induction of TAp73 by genotoxic stress requires Egr1 in mouse in vivo. Newly discovered non-consensus p53-binding sites in p73, p53 and Egr1 promoters reveal inter-regulating networks and sustained expression by feedback loops in response to stress, resulting in prolonged expression of the p53 family of genes and efficient apoptosis.

The heat shock response in HeLa cells is accompanied by elevated expression of the c-fos proto-oncogene.

Several known inducers of the heat shock response (heat stress, arsenite, and heavy metals) were shown to cause a significant elevation of c-fos mRNA in HeLa cells. Heat stress resulted in a time- and temperature-dependent prolonged elevation in the level of c-fos mRNA, which was accompanied by increased translation of c-fos protein and its appearance in the nucleus. Elevated expression of c-fos during heat stress was paralleled by induction of hsp 70 mRNA, while levels of c-myc and metallothionein mRNAs declined. Treatment of HeLa cells with arsenite or heavy metals also resulted in increased levels of hsp 70, as well as c-fos mRNA. Although elevated expression of c-fos was prevented by inhibitors of RNA synthesis, analysis of relative rates of gene transcription showed that during heat stress there was a negligible change in c-fos transcription. Therefore, the enhanced expression of c-fos during the heat shock response is likely to occur primarily through posttranscriptional processes. Cycloheximide was also shown to significantly increase the c-fos mRNA level in HeLa cells. There results are consistent with the observation that these inducers of the heat shock response, as well as cycloheximide, repress protein synthesis and suggest that the increase in the level of c-fos mRNA is caused by an inhibition of protein synthesis. This supports the hypothesis that c-fos mRNA is preferentially stabilized under conditions which induce the heat shock response, perhaps by decreased synthesis of a short-lived protein which regulates c-fos mRNA turnover.

Transcription of c-onc genes c-rasKi and c-fms during mouse development.

We investigated the expression of cellular sequences c-rasKi and c-fms, which are homologous to the oncogenes of Kirsten rat sarcoma virus and the McDonough strain of feline sarcoma virus, during murine development and in a variety of mouse tissues. The c-rasKi gene was found to be transcribed into two mRNA species of approximately 2.0 and 4.4 kilobases, whereas a single c-fms-related transcript of approximately 3.7 kilobases was identified. The c-rasKi gene appeared to be expressed ubiquitously, since similar levels of transcripts were observed in embryos, fetuses, extraembryonal structures, and a variety of postnatal tissues. In contrast, significant expression of c-fms was found to be confined to the placenta and extraembryonal membranes (i.e., combined yolk sac and amnion). The concentration of c-fms transcripts in the placenta increased approximately 15-fold (relative to day-7 to day-9 conceptuses) during development before reaching a plateau at day 14 to 15 of gestation. The time course of cfms expression in the extraembryonal membranes appeared to parallel the stage-specific pattern observed in the placenta. The level of c-fms transcripts in the extraembryonal tissues reached a level which was approximately 20- to 50-fold greater than that in the fetus. These findings suggest that the c-fms gene product may play a role in differentiation of extraembryonal structures or in transport processes occurring in these tissues. Our results indicate that the c-onc genes analyzed in the present study exert essentially different functions during mouse development.

The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin.

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFbeta1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.

Egr-1 negatively regulates human tumor cell growth via the DNA-binding domain.

Human HT1080 fibrosarcoma cells, subclone H4, express little or no Egr-1 (Zif/268, Krox 24), an early growth response gene encoding a transcription factor. Phorbol ester (but not serum) treatment only can elicit a small increase in Egr-1 expression in H4, in contrast to the normally rapid, high transient expression of Egr-1 observed after the addition of a wide range of stimulating agents to normal or immortalized cell lines. Because several human tumor cell lines express little Egr-1, we tested the hypothesis that this loss was causal to transformation. We report here that the expression of exogenous mouse Egr-1 in H4 cells inhibits transformed growth in a dose-dependent manner and significantly suppresses tumorigenicity in athymic mice. By overexpression of the fragment in Egr-1 that is responsible for its DNA-binding activity, the zinc-finger domain, we show that this domain has a similar activity. Moreover, the expression of antisense mRNA encoding the DNA-binding domain increases the transformed character of the H4 cells. One possible conclusion is that endogenous Egr-1-like genes perform growth-regulatory functions. Other human tumor lines are also growth suppressed by Egr-1 overexpression including ZR-75-1 breast carcinoma, U251 glioblastoma, and to a lesser extent, SAOS-2 osteosarcoma cells. These results are surprising in light of the "early growth response" character of Egr-1 but extend our earlier report of suppression of growth in v-sis-transformed NIH3T3 cells.

A biological role for Egr-1 in cell survival following ultra-violet irradiation.

The response to ultra-violet (u.v.) irradiation varies among cells, but commonly involves the rapid increase in expression of one or more transcription factors. The specific roles of this increased expression are largely unknown. We show here that in mouse NIH3T3 cells, Egr-1 expression is increased two-fold 10 min after u.v. irradiation, rises to a maximum (eightfold induction) after about 2 h and then declines. The expression of p53 protein is also strongly induced but is maximal between 2 to 4 h before declining. In contrast, the expression of c-Fos, and C-Jun proteins are only slightly affected by u.v. The Egr-1 response is independent of the growth state of the cells but depends on tyrosine kinase and protein kinase C activities. c-Ha-Ras is also involved in the induction of Egr-1 in u.v. irradiated cells. Evidence presented suggests that the mechanism for the response involves oxidative stress rather than DNA damage. We show that Egr-1 functions in the protection of cells against u.v. damage since NIH3T3 cells that constitutively express antisense Egr-1 and consequently cannot produce an Egr-1 response to u.v., grow at a rate 26% less than similarly irradiated parental cells and 36% less than nonirradiated parental cells. This is the second protective role described for Egr-1.

Suppression of v-sis-dependent transformation by the transcription factor, Egr-1.

The transcription factor Egr-1, stimulates the activity of a number of genes and inhibits other genes, by binding to the sequence GCGGGGGCG in 5' enhancer regions. However, the functions of Egr-1 are obscure in spite of its rather ubiquitous expression. Egr-1 may play a role in proliferation in mitogen-stimulated cells but its expression is also correlated with the differentiated state in teratocarcinoma cells. The constitutive expression of Egr-1 appears to have little effect on the growth rate of normal immortalized cell-lines. We show that in NIH3T3 cells that are conditionally transformed by the expression of v-six, the presence of Egr-1 is inhibitory to the production of transformed colonies (foci) and to growth in soft agar. In addition, the first appearance of tumors in nu/nu mice is delayed in tumorigenicity tests with cells that over-express Egr-1 and tumor growth is suppressed compared to control cells. We used a series of fragments of Egr-1 cloned into expression vectors to show that not only full length, but also truncated Egr-1 fragments inhibit colony formation. Using deletion mutants, we observed that this inhibitory activity is dependent on the presence of the DNA-binding 'zinc-finger' region. Wilm's tumor protein, WT1, (known to be a tumor suppressor gene) that exhibits the same DNA binding activity is also inhibitory. In contrast, colony formation is stimulated by an Egr-1 antisense RNA-expressing plasmid, since colonies grow rapidly and the colony-forming frequency is higher than in the presence of v-sis alone. We conclude that proteins containing the Egr-1 'zinc-finger' domain can bind to the regulatory regions of one or more genes that are required for the transformation of fibroblasts by v-sis thus inhibiting transformation. One function for Egr-1 implied by these results is the restraint of transformed growth in mitogen-stimulated cells.

Regulation of Egr-1 (Zfp-6) and c-fos expression in differentiating embryonal carcinoma cells.

The Egr-1 gene (zfp-6) encodes a 'zinc finger'-type transcription factor that is one of the early growth response genes induced, together with c-fos proto-oncogene, in many cell types. Our earlier work indicated that Egr-1 and c-fos may also play roles in differentiation and we now present data to show some features of their regulation. Transcriptional regulation accounts at least partly for the increased steady-state levels of Egr-1 mRNA in differentiating teratocarcinoma cells; this rate increases threefold over the 7-10 days of differentiation of P19 embryonal carcinoma cells with both 0.5% DMSO (to give predominantly cardiac muscle) and 1 microM retinoic acid (to give nerve and glial cells). The stability of Egr-1 transcripts remains the same (T1/2 = 90 min) in undifferentiated EC and differentiated cell products. In contrast, transcripts for c-fos are barely detectable in EC cells and increase 20-fold during differentiation. The basis for this is a marked increase in stability of c-fos mRNA after differentiation. The protein products of both genes parallel the steady-state levels of their mRNAs, but both proteins become more stable in differentiated cells. This is particularly marked for c-Fos protein, which appears as a distinct 58 kDa species in terminally differentiated P19 cells. Both Egr-1 and c-Fos proteins remain at high constitutive levels in differentiated cells indicating a distinct role for these transcription factors, For instance, it appears that this form of Fos protein may not repress the synthesis of the Egr-1 gene as it does during transient expression of serum-stimulated genes.

p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis.

The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.

Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines.

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.

Cripto: roles in mammary cell growth, survival, differentiation and transformation.

Cripto-1 (Cr-1) protein, encoded by the teratocarcinoma-derived growth factor gene (TDGF-1), is highly correlated with transformation in breast cancer. Eighty-two percent of breast carcinomas express Cr-1 whereas it is undetected in normal human breast tissue. We confirmed and extended findings that Cr-1 protein is expressed during the pregnancy and lactating stages of normal murine mammary glands but is barely detectable in glands from virgin animals and is undetectable in involuted glands. Cr-1 was found to be expressed in CID 9 cells, a line of mammary epithelial cells derived from 14.5 day pregnant mice and we have used these cells to investigate the roles of this gene. Exogenous mouse Cr-1 expression from a retroviral vector caused CID 9 cells to grow at an increased rate and to increased cell densities compared to parental and control cells. CID 9 cells overexpressing Cr-1 did not differentiate efficiently. Infection of CID 9 cells with a Cr-1 antisense vector caused these cells to change in morphology, to grow slowly, to undergo apoptosis at a higher rate and to achieve lower saturation densities but the cells were still capable of differentiating. We concluded that Cr-1 is an autocrine growth factor for normal breast cells, that when over-expressed stimulates excessive cell proliferation at the expense of differentiation. In transplantation studies, Cr-1 over-expression stimulated the growth and survival of mammary cells, but did not stimulate tumorigenesis in vivo.

Egr-1 inhibits apoptosis during the UV response: correlation of cell survival with Egr-1 phosphorylation.

UV irradiation of normal or immortalized cells induces a rapid increase in the expression of several transcription factors and is thought to serve a protective function. The human fibrosarcoma cell line, HT1080 clone H4, expresses almost undetectable levels of Egr-1 and does not respond to UV-C irradiation by the induction of Egr-1. The H4 cells are hypersensitive to UV which induces apoptosis and reduces clonogenicity. The introduction of exogenous Egr-1 into H4 (H4E9 and H4E4 cell-lines) confers protection from UV damage as measured by a number of assays. In both NIH3T3 (with inducible Egr-1) and H4E9 (constitutive Egr-1) cells, UV irradiation gave enhanced transactivation of Egr-1 reporters that correlated with phosphorylated Egr-1. Studies using inhibitors indicated that protein kinase-C and tyrosine kinases are involved in the anti-apoptotic effects of Egr-1 after UV damage. This is the first description of a biological effect of phosphorylated Egr-1.

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development.

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

Two proto-oncogenes that play dual roles in embryonal cell growth and differentiation.

The function of growth factors in early development is reviewed. Special emphasis is on the epidermal growth factor and its receptor, and on the c-fos and its family of transcriptional factor proteins, which play an important role in modulating the growth and differentiation of early embryos and embryonal carcinoma cells.